Reawakening a Sleeping Giant

The use of cells from two different species allows the team to distinguish events as the human X chromosome is reprogrammed, and some genes are turned on. Left panel show cells with both a human (red) and a mouse (green) nucleus, before these nuclei fuse. At this point, one human X chromosome is active (bright red dot). Then the second human X chromosome becomes reprogrammed (middle panel, two red dots). Finally, the nuclei of the two cells fuse (right panel) to form a hybrid cell with two active X chromosomes.
MRC Clinical Sciences Centre

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Reawakening a Sleeping Giant

Most molecular biologists look at how to switch on and regulate single genes. Scientists at the MRC Clinical Sciences Centre (CSC) have gone further, and have explored how to reawaken an entire set of inactive genes, a chromosome, that is present in every female human cell.

This reactivation happens when a ‘normal’ cell is turned back into a stem cell. The CSC team are the first to identify the earliest changes in this process. Understanding exactly how it happens could eventually help researchers to direct it, and produce stem cells tailored for use in therapies.

Stem cells found in early development have the ability to become any one of the many types of cell that make up our bodies. Differentiation into these cell types involves a series of “decisions” by the cell until it has only one role, for example being a skin cell. Scientists are interested in reversing these “decisions” to turn specialised cells back into their stem cell state. The goal is to be able to produce a pool of stem cells, which could then be directed to develop into any type of cell, and used to replace damaged or diseased tissue.

As a cell commits to a particular role, changes are made to its DNA so that genes no longer needed for that role can be retired from use. When scientists reverse this process, using a technique called ‘reprogramming’, these changes need to be undone so that the genes can be turned back on. The CSC team is the first to have identified the very earliest events that occur when ‘retired’ genes on the X chromosome are turned back on. The findings are published today in Nature Communications.

The DNA strands in each cell are organised into clusters, which are the chromosomes. There are two special chromosomes, called X and Y, which carry the information that determines sex. Every cell has two of these special chromosomes. Males have one X and one Y, while females have two Xs. Female cells only need one X, and using both would mean that an extra set of genes would be active. To avoid this, one chromosome gets randomly turned off in favour of the other. The CSC researchers explored how to turn the inactive X chromosome back on.

When the cell “chooses” an X chromosome to be turned off, it marks it with specific molecules. Some of these molecules bind to the DNA, which is wrapped up into a large coil, whilst others bind to proteins on the coil. These marks determine whether genes are turned on or not. They are called ‘epigenetic’ marks and are passed on to each cell’s “daughters” as it divides.

To reprogram a specialised cell back into a stem cell, scientists need to remove the epigenetic marks. If some of these marks remain, the stem cell will retain a tendency to make “decisions” that may lead it to become the same type of cell that it used to be. This limits its ability to become any type of cell in the body, and so limits its potential use in medical treatments.

“We don’t know exactly how to erase the previous memory, and this is extremely important if we want to use these cells again for therapy,” says Irene Cantone, of the CSC’s Lymphocyte Development group, and who helped to lead the research. The CSC team developed a technique that allowed them to watch what happened to an inactive X chromosome when it was woken up and readied for action. The technique involves fusing together a human female skin cell, which contains an inactive X chromosome, with a stem cell from a mouse embryo.

Fusing the cells together reprograms the skin cell towards a stem cell state. This happens because the mouse stem cell, unlike the human skin cell, contains all of the biological factors needed to reprogram a specialised cell. These factors invade the control centre, or nucleus, of the human cell and begin to adjust the epigenetic marks, allowing genes that had been retired to start afresh. The researchers constructed a timeline of these epigenetic changes. “I now have a better idea of what is needed for these genes to be reactivated,” says Cantone.

A pivotal moment is when the two nuclei fuse together. By observing the changes that take place before and after the nuclei fuse, scientists can begin to work out which cellular mechanisms play a role in reprogramming the cell and reactivating the dormant X chromosome.

Cantone and colleagues have shown that two molecules, called XIST (X-inactive specific transcript) and H3K27me3, play a key role in events before the nuclei fuse. The normal role of these molecules in a skin cell is to help silence the inactive X chromosome. They coat the DNA to prevent the cellular machinery from accessing certain genes, and in doing so turn them off. The researchers showed that when the skin cell begins to be reprogrammed, these markers are lost or move away from the chromosome before genes are turned back on.

Not all genes on the silent X chromosome are woken up during this process. “What we found is that only some genes are re-activated, and many stay silent. We now need to know what is the basis of this difference. Why are some sensitive, and others not?” says Amanda Fisher, also of the CSC’s Lymphocyte Development group, and a lead scientist on the study.
If scientists can understand how to reverse the biological process of gene silencing that exists inside cells, they may one day be able to produce stem cells suitable to replace damaged and diseased tissue.

The results also have relevance to diseases linked to the X chromosome, such as Duchenne muscular dystrophy, red-green colour blindness and Rett syndrome. “If we can understand how to reactivate specific genes on an inactive chromosome and in certain cells, this could lead to improved treatments in the future,” says Cantone.